Estradiol levels monitored 78 to 117 days after transplantation.
A, Levels from the RH (right hand) show 2 surges. B, Levels from the RCF (right
cubital fossa) show larger fluctuations, and are comparable with ovarian vein
measurements.

Figure 4. Oocyte Retrieval From the Subcutaneously
Grafted Ovary in Patient A

A, Metaphase I oocyte retrieved from a 14-mm follicle. B, Same oocyte
after in vitro maturation to metaphase stage II. Note the extruded polar body.
Bright field microscopy with Hoffman modulation contrast using a Nikon Diaphot
Microscope.

Table. Hormone Measurements 78 to 140 Days
After Transplantation Comparing Peripheral and Central Values in Patient A

Context In reproductive-age women, one of the common adverse effects of chemotherapy
and radiotherapy is premature ovarian failure. In addition, a significant
number of women experience early menopause due to oophorectomy performed for
benign indications.

Design and Setting Case study of 2 patients in New York who received autologous ovarian
transplantation (patient A, November 1999; patient B, April 2000) to the forearm
prior to pelvic radiotherapy or after oophorectomy.

Main Outcome Measures Follicular development evident by ultrasound examination; cyclical production
of estradiol and progesterone; restoration of serum follicle-stimulating hormone,
luteinizing hormone, and testosterone levels to nonmenopausal range; and disappearance
of menopausal symptoms.

In women, one of the common and distressing adverse effects of chemotherapy
and pelvic radiotherapy is premature ovarian failure.1
Previously, ovarian transposition was performed with varying degrees of success
but the scatter radiation and vascular compromise limited its effectiveness.2,3 Moreover, this treatment could not
protect against the gonadotoxicity of chemotherapy. Recently, a multitude
of laboratory, animal, and human xenograft studies has described an emerging
alternative for these patients: autotransplantation of fresh or frozen-banked
ovarian cortical strips.4 In addition, many
women experience premature menopause due to oophorectomies performed for benign
ovarian cysts or endometriosis. Ovarian autotransplantation may restore ovarian
function in these patients.5,6

We have reported the first case of laparoscopic transplantation of frozen-thawed
ovarian tissue to the pelvic sidewall with subsequent ovulation.5,6
However, when there is a possibility that a patient may receive pelvic radiotherapy,
or when close surveillance of ovarian tissue is required, a heterotopic location
may be more desirable. We chose the forearm, based on previous studies demonstrating
that both fresh and frozen-thawed parathyroid tissues have been successfully
transplanted in this location.7,8

METHODS

The study was approved by the institutional review board committees
at the New York Methodist and New York Presbyterian hospitals.

Patient A

A 35-year-old woman was diagnosed with stage IIIB squamous cell cervical
carcinoma. She consented to fresh transplantation prior to pelvic radiotherapy
to preserve her ovarian function. After frozen section biopsies showed no
metastasis, both ovaries were removed laparoscopically, and their cortices
were prepared in 16 strips of 5 × 50 × 1 to 3 mm.9
A 1-cm vertical incision was made over the brachioradialis muscle, 5 cm below
the antecubital fossa. Ovarian strips were wedged subcutaneously, using a
suture pull-through technique. The patient was started on 1 mg of micronized
estradiol on postoperative day 2.

The patient was radiosensitized with cisplatinum (50 mg/m2
× 1) and 5-fluorouracil (1000 mg/m2 per day on days 3-6).
This course was repeated 4 weeks later. During weeks 1 through 5, the patient
received external beam radiotherapy to the pelvis for a total dose of 5040
cGy. During this radiotherapy, the patient's arm was raised above her head
and shielded. Two weeks later, 2 sessions of brachytherapy were performed
with cesium.

Patient B

A 37-year-old woman developed a recurrent benign serous cyst in her
only ovary. Her 1 ovary had been previously removed due to a serous cystadenoma.
The patient had a "frozen-pelvis" due to dense pelvic adhesions and she has
had multiple laparotomies for cystectomies on the remaining ovary. After the
third recurrence, a decision was made by her gynecologist to perform an oophorectomy.
After the removal of the ovary, healthy ovarian tissue was harvested from
the specimen. The transplantation technique was similar to that in the first
patient, but for aesthetic reasons the tissue was transplanted more medially
in the forearm.

RESULTS

Patient A

Six weeks after the transplantation, the patient's follicle-stimulating
hormone (FSH) and luteinizing hormone (LH) were 47 and 35 mIU/mL, respectively.
Approximately 10 weeks after the transplant, she reported the presence of
a painless bulge at the site of the transplant (Figure 1). Ultrasound examination showed a 15-mm dominant follicle
(Figure 2A) and 4 other antral follicles,
measuring 5 to 7 mm (Figure 2B),
at which point estrogen replacement was discontinued. Repeat hormonal analyses
showed estrogen production, as well as normalization of FSH and LH concentrations.
The testosterone levels were also restored to the nonmenopausal range (Table 1). Serial ultrasound examinations
showed continual development of new antral follicles as large as 15.5 mm.

To determine the cyclical hormone production, the patient was monitored
every 1 to 5 days during postoperative days 78 through 117. The right hand
(RH) estradiol measurements representing peripheral hormone levels showed
2 surges (Figure 3A). Estradiol
levels from the right cubital fossa (RCF), which represent the immediate output
of the graft, showed a significantly higher average (Figure 3B) compared with the peripheral levels (RH). Although serum
progesterone levels fluctuated between 0.13 and 0.38 ng/mL, they did not reach
ovulatory levels.

Table 1 reflects extended
monitoring between 78 and 140 days after the transplantation. There was a
statistically significant gradient in estradiol, progesterone, and testosterone
between the RH and RCF measurements. The values from the RCF resembled those
of the ovarian vein,10 indicating that the
veins in that area had assumed ovarian drainage.

Because the longevity of grafted tissue could not be predicted, the
patient wished to cryopreserve embryos for future use with a gestational surrogate.
A percutaneous oocyte retrieval was performed on postoperative day 216, when
the RH estradiol level was 157 pg/mL (576 pmol/L). An immature oocyte containing
a germinal vesicle was obtained from an 11-mm follicle.

To synchronize follicle development in the subsequent attempt, the patient's
pituitary gonadotropin production was blocked with a gonadotropin-releasing
hormone antagonist (250 µg/d of Antagon for 2 days) (Serono, Norwell,
Mass). Ovarian stimulation was performed with 150 to 225 IU of human menopausal
gonadotropins (Pergonal; Serono) and 150 to 300 IU of recombinant FSH (Follistim;
Organon Inc, West Orange, NJ). After 11 days of stimulation, 4 follicles ranging
in size from 11.5 to 15.5 mm were visualized on ultrasound. The estradiol
level for RCF was 3482 pg/mL (12 782 pmol/L) and was 264 pg/mL (969 pmol/L)
for RH.

Thirty-six hours after the administration of human chorionic gonadotropin,
3 oocytes were recovered percutaneously. Two oocytes from 15.5-mm follicles
were postmature; an 11.5-mm follicle did not yield an oocyte; and an oocyte
from a 14-mm follicle was in metaphase I (Figure 4A). This oocyte was matured in vitro overnight (Figure 4B); intracytoplasmic sperm injection
with donor sperm did not result in fertilization.

In response to the human chorionic gonadotropin injection, the patient's
RH and RCF progesterone levels reached mean (SE) ovulatory levels of 3.08
(1.43) and 19.2 (12), respectively). Culture of granulosa cells from the 14-mm
follicle confirmed progesterone production in vitro (data not shown).

Approximately 10 months after the transplantation, the patient was diagnosed
with local recurrence in the pelvic sidewall and was treated with 3 courses
of 650 mg of carboplatinum over 9 weeks. Despite this treatment, the patient's
FSH levels remained near the normal mean (SE) of 10.2 (1.5) mIU/mL at the
18-month follow-up.

Patient B

A postoperative FSH level of 50.7 mIU/mL confirmed menopause. Five months
later, the patient felt a lump growing at the transplant site. One month later,
an ultrasound showed a 7.5-mm follicle in the forearm, which grew to 9 mm
in 2 days. Hormone replacement (1 mg of micronized estradiol and 10 mg of
medroxyprogesterone acetate) was discontinued. During the subsequent month
the patient reported spontaneous menstruation. On day 13 of that cycle, a
9-mm follicle was noted by ultrasound and her hormone measurements indicated
a mid-cycle surge (FSH, 40 mIU/mL; LH, 90 mIU/mL; estradiol, 254 pg/mL [932
pmol/L]; progesterone, 2.1 ng/mL). The patient menstruated spontaneously 2
weeks later, and every 25 to 28 days thereafter. Ten months after the transplant
the patient was monitored again. Six to 11 days after an LH surge (62 mIU/mL),
progesterone ranged from 7 to 10.1 ng/mL (mean [SE], 8.5 [0.9] ng/mL), confirming
spontaneous ovulation. Levels of FSH, LH, and estradiol on the second day
of menstruation indicated normal ovarian reserve (15.4 mIU/mL, 6.6 mIU/mL,
and 47 pg/mL, respectively).11 Probably because
the tissue was transplanted more medially, no gradient was noted between the
RH and RCF.

Neither patient complained of discomfort, but indicated that they could
"feel" the follicle during mid cycle. Their moliminal symptoms returned and
both expressed a sense of well-being with endogenous estrogen. Patient A preferred
to wear long-sleeve shirts mid cycle, while patient B did not have any cosmetic
concerns.

COMMENT

This is the first report of endocrine function and oocyte retrieval
after autologous grafting of ovarian cortical strips to the forearm. This
implantation site affords easy access for grafting, monitoring, and ultrasound
examination.

The lack of spontaneous ovulation in patient A can be attributed to
the fact that estradiol levels never exceeded 250 pg/mL (918 pmol/L), which
is a prerequisite for induction of an LH surge.12
However, in vivo and in vitro progesterone production in response to human
chorionic gonadotropin indicated that the granulosa cells were capable of
luteinization. In patient B, peak estradiol levels exceeded 250 pg/mL (918
pmol/L) and spontaneous ovulation and menstruation occurred. A recent primate
study also showed restoration of spontaneous ovulation after transplantation
of fresh and frozen-thawed strips to the forearm.13

Ovarian endocrine function was restored despite the radiosensitizing
chemotherapy. This can be explained by the fact that the dose was small and
because relatively nongonadotoxic drugs were used compared with the alkylating
agents. In addition, while the revascularization was in progress during the
first week, tissue diffusion of these drugs was probably restricted. Exposure
to chemotherapy, however, could be completely avoided by ovarian cryopreservation,
and by delaying the transplantation after the treatment. In the present cases,
this could not be done because the age limit for cryopreservation was set
at 34 years in the research protocol.

In conclusion, ovarian transplantation to the forearm results in endocrine
function. With optimization of ovarian stimulation and percutaneous oocyte
retrieval techniques, this procedure may also restore fertility in the near
future when used in conjunction with in vitro fertilization.